Temporal and spatial control of intracellular Ca2+ fluctuations are of critical importance for eukaryotic cell function. The long-term research goal underlying this proposal is to understand the regulation of Ca2+ extrusion by the plasma membrane Ca2+ ATPase (PMCA) as the primary high affinity calcium efflux system of eukaryotic cells. Over twenty PMCA isoforms are generated from a multigene family and via alternative RNA splicing. The complex pattern of tissue and cellular distribution of these isoforms suggests their functional adaptation to the specific needs of a given cell. This project will explore the hypothesis that specific plasma membrane Ca2+ ATPase isoforms are targeted to multiprotein complexes where they associate with other receptors, transporters, channels, and signaling molecules to form "units of local Ca2+ regulation" at specific membrane sites. This concept has recently gained experimental support by the demonstration that some PMCA isoforms interact via their C-terminal tails with PDZ (PSD-95/Dlg/ZO-1) protein-protein interaction domains present in a growing number of multi-modular proteins thought to cluster and anchor membrane transporters and signaling proteins at the plasma membrane. The specific aims are (1) to determine the effect of PDZ domain interaction on the function of PMCAs; (2) to identify and characterize bona fide PDZ domain proteins that interact with different PMCA isoforms; and (3) to determine the cellular localization and possible co-localization of PMCA isoforms with candidate interacting PDZ domain proteins. Methods will include functional Ca2+ uptake assays, yeast two-hybrid screenings and biochemical, molecular and immunological techniques to identify and characterize specific protein-protein interactions; as well as confocal light microscopy combined with protein overexpression and fluorescent labeling methods to determine specific protein localizations. These experiments will open a new area for investigations of the physiological role of Ca2+ ATPase isoforms in the local control of Ca2+ regulation at the plasma membrane. For the plasma membrane Ca2+ ATPases, this represents a paradigm shift, changing their static image as housekeeping system responsible for the global maintenance of intracellular Ca2+ levels to one of a dynamic component involved in local Ca2+ control and Ca2+ signal transduction.